This invention relates to immunogenic epitopes of the human zona pellucida protein (ZP1) and their use as contraceptive agents. Particularly, the invention provides immunogenic ZP1 epitopes useful for inducing anti-zona pellucida antibodies in multiple mammalian species.
The zona pellucida (ZP) is the glycoprotein extracellular matrix formed during mammalian ovarian follicular development (1-6). The proteins of this ZP are produced by the oocyte as well as by granulosa cells. The zona pellucida proteins are involved in several critical stages of the fertilization process, including: attachment, binding, and penetration by the capacitated spermatozoa; induction of the acrosome reaction and prevention of polyspermy (7-9).
While it has long been noted that the mammalian ZP is composed of three major glycoproteins, the classification of these proteins has been complicated, in part due to extensive species variation in post-translational modification of the ZP proteins, including glycosylation and sulfation. Because of these modifications, the proteins exhibit extensive heterogeneity. Therefore, the nomenclature, based on electrophoretic mobilities of the ZP proteins of different species, has been difficult. As the cDNAs and genes encoding the different ZP proteins have been isolated, it has only recently been possible to distinguish the ZP gene families (9-15, 17-21). For clarification of ZP terms used in this study, a summary of nomenclature of these gene families as they relate to the mouse ZP nomenclature is provided in Table 1.
Although the genes encoding the ZP proteins are evolutionarily conserved, there appear to be major differences in these proteins relative to their immunochemical and functional properties (16, 22). In the mouse, ZP3 is the primary spermatozoa binding ZP protein (23) while N-linked but not 0-linked carbohydrates are implicated in sperm binding to porcine ZP (24, 25). However, the rabbit ZP1 (55 kDa) and pig ZP1 (also referred to as ZP3alpha), which are homologues of the mouse ZP1, but not the mouse ZP3 family, exhibit spermoatozoa receptor activity (16, 26).
Recent studies suggest that interactions between the ZP protein and sperm is more complicated than that proposed for the mouse model. Both protein and carbohydrate moieties appear to be involved in spermatozoa binding in pigs (25-27) and multiple ZP proteins may be involved in this interaction in pigs and rabbits. Regardless of the precise molecular mechanisms of sperm interaction with the ZP, the efficacy of immunization with zona pellucida proteins for contraception ultimately depends upon the production of sufficient antibody titers to inhibit fertilization at any stage of the fertilization process.
The immunogenicity and antigenicity of ZP proteins is complex (9, 13, 22, 28). To date, most of the studies to identify specific peptide epitopes have used monoclonal antibodies made in mice (14, 29-32). It has been clearly established that the immunogenicity of non-rodent ZP proteins injected into mice is distinct from that in other mammals and that immunization of mice is distinct from immunization of other mammals. Importantly, immunization of mice and rats with porcine ZP does not effect fertility (33, 34). It is critical to demonstrate the immune response in non-rodent models, for example primate models, to design an effective and safe human contraceptive vaccine.
To date, primate models that have been used in such studies include: baboons (Papio anuhis) (35); squirrel monkeys (Saimiri sciureus) (36), marmosets (Callithrix jacchus) (37) and cynomolgous macaques (Macaca fascicularis) (1 6, 38, 39). Aitken and colleagues (40) have demonstrated that immunization of marmosets with native porcine ZP3 and recombinant cDNA expressed ZP3 protein interferes with normal ovarian function, evidenced by histological analysis and fertility outcomes.
Studies by Dunbar and colleagues in the cynomolgous monkey model, demonstrate that immunization with recombinant rabbit 55 kDa ZP protein (ZP1 protein family) conjugated to Protein A produces a significant humoral immune response when used with the muramyl depeptide (38, 41). These monkeys retain normal endocrine function as well as ovarian morphology. However, as importantly, the antibodies developed by these animals inhibited monkey sperm from binding to homologous monkey ZP.
Given the significance of these observations and the potential for the use of the ZP1 protein as a safe but effective immunocontraceptive agent in humans, it would be useful to identify immunodominant B cell linear peptide epitopes of the human ZP1 protein that could be used, for example, in a contraceptive vaccine.
The invention provides immunogenic epitopes of the zona pellucida protein (ZP 1), preferably an immunogenic ZP1 amino acid sequence of 30 or fewer amino acids. Immunogenic epitopes of the invention include those having the following amino acid sequence: GPLX1X2X3LX4I [SEQ ID NO: 1], where X1 is T or R, X2 is L or V, X3 is E or V, and X4 is Q or R. Particularly useful peptide epitopes are:
GPLTLELQI [SEQ. ID NO: 2],
GPLTVVLQI [SEQ. ID NO: 97], and
GPLRLELRI [SEQ. ID NO: 98]
The immunogenic ZP epitopes of the invention are recognized by antibodies raised against two or more differing mammalian species ZP antigens, demonstrating their value as contraceptive agents. When administered to a mammal; the amino acid sequences of the invention induce production of anti-ZP antibodies. The production of anti-ZP antibodies interferes with fertilization and/or contraception.